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1.
J Neurosci ; 44(27)2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38692734

RESUMO

Aberrant condensation and localization of the RNA-binding protein (RBP) fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Changes in RBP function are commonly associated with changes in axonal cytoskeletal organization and branching in neurodevelopmental disorders. Here, we asked whether branching defects also occur in vivo in a model of FUS-associated disease. We use two reported Xenopus models of ALS/FTD (of either sex), the ALS-associated mutant FUS(P525L) and a mimic of hypomethylated FUS, FUS(16R). Both mutants strongly reduced axonal complexity in vivo. We also observed an axon looping defect for FUS(P525L) in the target area, which presumably arises due to errors in stop cue signaling. To assess whether the loss of axon complexity also had a cue-independent component, we assessed axonal cytoskeletal integrity in vitro. Using a novel combination of fluorescence and atomic force microscopy, we found that mutant FUS reduced actin density in the growth cone, altering its mechanical properties. Therefore, FUS mutants may induce defects during early axonal development.


Assuntos
Esclerose Lateral Amiotrófica , Axônios , Demência Frontotemporal , Mutação , Proteína FUS de Ligação a RNA , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Axônios/patologia , Axônios/metabolismo , Animais , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Demência Frontotemporal/metabolismo , Feminino , Masculino , Xenopus laevis , Cones de Crescimento/metabolismo , Humanos , Modelos Animais de Doenças
2.
Sci Adv ; 10(22): eadn2050, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38809982

RESUMO

Transporting and translating mRNAs in axons is crucial for neuronal viability. Local synthesis of nuclear-encoded mitochondrial proteins protects long-lived axonal mitochondria from damage; however, the regulatory factors involved are largely unknown. We show that CLUH, which binds mRNAs encoding mitochondrial proteins, prevents peripheral neuropathy and motor deficits in the mouse. CLUH is enriched in the growth cone of developing spinal motoneurons and is required for their growth. The lack of CLUH affects the abundance of target mRNAs and the corresponding mitochondrial proteins more prominently in axons, leading to ATP deficits in the growth cone. CLUH interacts with ribosomal subunits, translation initiation, and ribosome recycling components and preserves axonal translation. Overexpression of the ribosome recycling factor ABCE1 rescues the mRNA and translation defects, as well as the growth cone size, in CLUH-deficient motoneurons. Thus, we demonstrate a role for CLUH in mitochondrial quality control and translational regulation in axons, which is essential for their development and long-term integrity and function.


Assuntos
Axônios , Mitocôndrias , Neurônios Motores , Doenças do Sistema Nervoso Periférico , Biossíntese de Proteínas , Animais , Neurônios Motores/metabolismo , Mitocôndrias/metabolismo , Axônios/metabolismo , Camundongos , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/patologia , Cones de Crescimento/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Camundongos Knockout
3.
PLoS One ; 19(5): e0295701, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38771761

RESUMO

The Polarity/Protusion model of UNC-6/Netrin function in axon repulsion does not rely on a gradient of UNC-6/Netrin. Instead, the UNC-5 receptor polarizes the VD growth cone such that filopodial protrusions are biased to the dorsal leading edge. UNC-5 then inhibits growth cone protrusion ventrally based upon this polarity, resulting in dorsally-biased protrusion and dorsal migration away from UNC-6/Netrin. While previous studies have shown that UNC-5 inhibits growth cone protrusion by destabilizing actin, preventing microtubule + end entry, and preventing vesicle fusion, the signaling pathways involved are unclear. The SRC-1 tyrosine kinase has been previously shown to physically interact with and phosphorylate UNC-5, and to act with UNC-5 in axon guidance and cell migration. Here, the role of SRC-1 in VD growth cone polarity and protrusion is investigated. A precise deletion of src-1 was generated, and mutants displayed unpolarized growth cones with increased size, similar to unc-5 mutants. Transgenic expression of src-1(+) in VD/DD neurons resulted in smaller growth cones, and rescued growth cone polarity defects of src-1 mutants, indicating cell-autonomous function. Transgenic expression of a putative kinase-dead src-1(D831A) mutant caused a phenotype similar to src-1 loss-of-function, suggesting that this is a dominant negative mutation. The D381A mutation was introduced into the endogenous src-1 gene by genome editing, which also had a dominant-negative effect. Genetic interactions of src-1 and unc-5 suggest they act in the same pathway on growth cone polarity and protrusion, but might have overlapping, parallel functions in other aspects of axon guidance. src-1 function was not required for the effects of activated myr::unc-5, suggesting that SRC-1 might be involved in UNC-5 dimerization and activation by UNC-6, of which myr::unc-5 is independent. In sum, these results show that SRC-1 acts with UNC-5 in growth cone polarity and inhibition of protrusion.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Polaridade Celular , Cones de Crescimento , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Movimento Celular , Cones de Crescimento/metabolismo , Receptores de Netrina/metabolismo , Receptores de Netrina/genética , Netrinas , Receptores de Superfície Celular
4.
Curr Opin Cell Biol ; 88: 102358, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38608424

RESUMO

Recent advances in imaging methods begin to further illuminate the inner workings of neurons. Views of the axonal landscape through the lens of in situ cryo-electron tomography (cryo-ET) provide a high-resolution atlas of the macromolecular organization in near-native conditions, leading to our growing understanding of the vital roles of compositional and structural organization in maintaining neuronal homeostasis. In this review, we discuss the latest observations concerning the fundamental components found within neuronal compartments, with special emphasis on the axon, branch points, and growth cone. We describe the similarity and difference in organization of organelles and molecules in varying compartments. Finally, we highlight outstanding questions on the dynamics and localization of various components along the axon that may be answered using orthogonal approaches.


Assuntos
Axônios , Axônios/metabolismo , Animais , Humanos , Microscopia Crioeletrônica , Cones de Crescimento/metabolismo
5.
Methods Mol Biol ; 2794: 177-186, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38630229

RESUMO

Immunocytochemistry combined with confocal or superresolution microscopy allows us to observe molecular localization and intracellular structures. However, it is challenging to analyze individual neurons in brain tissue, where neurons are densely packed. In contrast, we can easily observe structures such as the axonal growth cone and dendritic spines in dissociated individual neurons. Thus, the immunocytochemistry of primary cultured neurons is often used because it reflects the in vivo condition at least in part. Here, we describe a method for indirect fluorescence immunocytochemistry of primary cultured neurons from the embryonic cerebral cortex. This involves multiple steps including fixation, permeabilization, and antibody reaction, and in particular, we introduce an optimized protocol for permeabilization to enable the precise localization of target molecules.


Assuntos
Anticorpos , Córtex Cerebral , Imuno-Histoquímica , Cones de Crescimento , Neurônios
6.
J Neural Eng ; 21(2)2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38547528

RESUMO

Objective. Cochlear implants provide auditory perception to those with severe to profound sensorineural hearing loss: however, the quality of sound perceived by users does not approximate natural hearing. This limitation is due in part to the large physical gap between the stimulating electrodes and their target neurons. Therefore, directing the controlled outgrowth of processes from spiral ganglion neurons (SGNs) into close proximity to the electrode array could provide significantly increased hearing function.Approach.For this objective to be properly designed and implemented, the ability and limits of SGN neurites to be guided must first be determined. In this work, we engineer precise topographical microfeatures with angle turn challenges of various geometries to study SGN pathfinding and use live imaging to better understand how neurite growth is guided by these cues.Main Results.We find that the geometry of the angled microfeatures determines the ability of neurites to navigate the angled microfeature turns. SGN neurite pathfinding fidelity is increased by 20%-70% through minor increases in microfeature amplitude (depth) and by 25% if the angle of the patterned turn is made obtuse. Further, we see that dorsal root ganglion neuron growth cones change their morphology and migration to become more elongated within microfeatures. Our observations also indicate complexities in studying neurite turning. First, as the growth cone pathfinds in response to the various cues, the associated neurite often reorients across the angle topographical microfeatures. Additionally, neurite branching is observed in response to topographical guidance cues, most frequently when turning decisions are most uncertain.Significance.Overall, the multi-angle channel micropatterned substrate is a versatile and efficient system to assess neurite turning and pathfinding in response to topographical cues. These findings represent fundamental principles of neurite pathfinding that will be essential to consider for the design of 3D systems aiming to guide neurite growthin vivo.


Assuntos
Implantes Cocleares , Neuritos , Cones de Crescimento , Células Cultivadas , Neurônios , Gânglio Espiral da Cóclea
7.
Science ; 383(6687): 1084-1092, 2024 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-38452066

RESUMO

The idea of guidance toward a target is central to axon pathfinding and brain wiring in general. In this work, we show how several thousand axonal growth cones self-pattern without target-dependent guidance during neural superposition wiring in Drosophila. Ablation of all target lamina neurons or loss of target adhesion prevents the stabilization but not the development of the pattern. Intravital imaging at the spatiotemporal resolution of growth cone dynamics in intact pupae and data-driven dynamics simulations reveal a mechanism by which >30,000 filopodia do not explore potential targets, but instead simultaneously generate and navigate a dynamic filopodial meshwork that steers growth directions. Hence, a guidance mechanism can emerge from the interactions of the axons being guided, suggesting self-organization as a more general feature of brain wiring.


Assuntos
Orientação de Axônios , Drosophila melanogaster , Cones de Crescimento , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Cones de Crescimento/fisiologia , Neurônios/fisiologia , Pseudópodes/fisiologia
8.
Nat Commun ; 15(1): 1877, 2024 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-38461182

RESUMO

Axonal growth cones mediate axonal guidance and growth regulation. We show that migrating neurons in mice possess a growth cone at the tip of their leading process, similar to that of axons, in terms of the cytoskeletal dynamics and functional responsivity through protein tyrosine phosphatase receptor type sigma (PTPσ). Migrating-neuron growth cones respond to chondroitin sulfate (CS) through PTPσ and collapse, which leads to inhibition of neuronal migration. In the presence of CS, the growth cones can revert to their extended morphology when their leading filopodia interact with heparan sulfate (HS), thus re-enabling neuronal migration. Implantation of an HS-containing biomaterial in the CS-rich injured cortex promotes the extension of the growth cone and improve the migration and regeneration of neurons, thereby enabling functional recovery. Thus, the growth cone of migrating neurons is responsive to extracellular environments and acts as a primary regulator of neuronal migration.


Assuntos
Cones de Crescimento , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores , Camundongos , Animais , Cones de Crescimento/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Neurogênese , Axônios/metabolismo , Sulfatos de Condroitina/metabolismo , Encéfalo/metabolismo , Células Cultivadas
9.
Mol Biol Cell ; 35(4): ar47, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38354034

RESUMO

Neuronal growth cones sense a variety of cues including chemical and mechanical ones to establish functional connections during nervous system development. Substrate-cytoskeletal coupling is an established model for adhesion-mediated growth cone advance; however, the detailed molecular and biophysical mechanisms underlying the mechanosensing and mechanotransduction process remain unclear. Here, we adapted a motor-clutch model to better understand the changes in clutch and cytoskeletal dynamics, traction forces, and substrate deformation when a growth cone interacts with adhesive substrates of different stiffnesses. Model parameters were optimized using experimental data from Aplysia growth cones probed with force-calibrated glass microneedles. We included a reinforcement mechanism at both motor and clutch level. Furthermore, we added a threshold for retrograde F-actin flow that indicates when the growth cone is strongly coupled to the substrate. Our modeling results are in strong agreement with experimental data with respect to the substrate deformation and the latency time after which substrate-cytoskeletal coupling is strong enough for the growth cone to advance. Our simulations show that it takes the shortest time to achieve strong coupling when substrate stiffness was low at 4 pN/nm. Taken together, these results suggest that Aplysia growth cones respond faster and more efficiently to soft than stiff substrates.


Assuntos
Cones de Crescimento , Mecanotransdução Celular , Cones de Crescimento/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Células Fotorreceptoras Retinianas Cones
10.
J Cell Sci ; 137(1)2024 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-38197773

RESUMO

Direct binding of netrin receptors with dynamic microtubules (MTs) in the neuronal growth cone plays an important role in netrin-mediated axon guidance. However, how netrin-1 (NTN1) regulates MT dynamics in axon turning remains a major unanswered question. Here, we show that the coupling of netrin-1 receptor DCC with tau (MAPT)-regulated MTs is involved in netrin-1-promoted axon attraction. Tau directly interacts with DCC and partially overlaps with DCC in the growth cone of primary neurons. Netrin-1 induces this interaction and the colocalization of DCC and tau in the growth cone. The netrin-1-induced interaction of tau with DCC relies on MT dynamics and TUBB3, a highly dynamic ß-tubulin isotype in developing neurons. Netrin-1 increased cosedimentation of DCC with tau and TUBB3 in MTs, and knockdown of either tau or TUBB3 mutually blocked this effect. Downregulation of endogenous tau levels by tau shRNAs inhibited netrin-1-induced axon outgrowth, branching and commissural axon attraction in vitro, and led to defects in spinal commissural axon projection in vivo. These findings suggest that tau is a key MT-associated protein coupling DCC with MT dynamics in netrin-1-promoted axon attraction.


Assuntos
Axônios , Cones de Crescimento , Netrina-1 , Neurônios , Microtúbulos
11.
Biol Chem ; 405(1): 31-41, 2024 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-37950644

RESUMO

Growth cones of oligodendrocyte progenitor cells (OPCs) are challenging to investigate with conventional light microscopy due to their small size. Especially substructures such as filopodia, lamellipodia and their underlying cytoskeleton are difficult to resolve with diffraction limited microscopy. Light microscopy techniques, which surpass the diffraction limit such as stimulated emission depletion microscopy, often require expensive setups and specially trained personnel rendering them inaccessible to smaller research groups. Lately, the invention of expansion microscopy (ExM) has enabled super-resolution imaging with any light microscope without the need for additional equipment. Apart from the necessary resolution, investigating OPC growth cones comes with another challenge: Imaging the topography of membranes, especially label- and contact-free, is only possible with very few microscopy techniques one of them being scanning ion conductance microscopy (SICM). We here present a new imaging workflow combining SICM and ExM, which enables the visualization of OPC growth cone nanostructures. We correlated SICM recordings and ExM images of OPC growth cones captured with a conventional widefield microscope. This enabled the visualization of the growth cones' membrane topography as well as their underlying actin and tubulin cytoskeleton.


Assuntos
Microscopia , Células Precursoras de Oligodendrócitos , Microscopia/métodos , Cones de Crescimento , Citoesqueleto , Microtúbulos
12.
Biol Pharm Bull ; 46(12): 1820-1825, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38044101

RESUMO

The polarity of the biological membrane, or lipid order, regulates many cellular events. It is generally believed that the plasma membrane polarity is regulated according to cell type and function, sometimes even within a cell. Neurons have a variety of functionally specialized subregions, each of which bears distinct proteins and lipids, and the membrane polarity of the subregions may differ accordingly. However, no direct experimental evidence of it has been presented to date. In the present study, we used a cell-impermeable solvatochromic membrane probe NR12A to investigate the local polarity of the plasma membrane of neurons. Both in hippocampal and cerebellar granule neurons, growth cones have higher membrane polarity than the cell body. In addition, the overall variation in the polarity value of each pixel was greater in the growth cone than in cell bodies, suggesting that the lateral diffusion and/or dynamics of the growth cone membrane are greater than other parts of the neuron. These tendencies were much less notably observed in the lamellipodia of a non-neuronal cell. Our results suggest that the membrane polarity of neuronal growth cones is unique and this characteristic may be important for its structure and function.


Assuntos
Corpo Celular , Cones de Crescimento , Neurônios/metabolismo , Membrana Celular , Hipocampo , Células Cultivadas
13.
Development ; 150(19)2023 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-37747104

RESUMO

During neural circuit formation, axons navigate from one intermediate target to the next, until they reach their final target. At intermediate targets, axons switch from being attracted to being repelled by changing the guidance receptors on the growth cone surface. For smooth navigation of the intermediate target and the continuation of their journey, the switch in receptor expression has to be orchestrated in a precisely timed manner. As an alternative to changes in expression, receptor function could be regulated by phosphorylation of receptors or components of signaling pathways. We identified Cables1 as a linker between floor-plate exit of commissural axons, regulated by Slit/Robo signaling, and the rostral turn of post-crossing axons, regulated by Wnt/Frizzled signaling. Cables1 localizes ß-catenin, phosphorylated at tyrosine 489 by Abelson kinase, to the distal axon, which in turn is necessary for the correct navigation of post-crossing commissural axons in the developing chicken spinal cord.


Assuntos
Orientação de Axônios , Axônios , Orientação de Axônios/fisiologia , Axônios/metabolismo , Cones de Crescimento , Medula Espinal/metabolismo , Via de Sinalização Wnt , Animais , Galinhas
14.
Int J Mol Sci ; 24(18)2023 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-37762659

RESUMO

Axon regeneration is essential for successful recovery after peripheral nerve injury. Although growth cone reformation and axonal extension are crucial steps in axonal regeneration, the regulatory mechanisms underlying these dynamic processes are poorly understood. Here, we identify ßPix (Arhgef7), the guanine nucleotide exchange factor for Rac1 GTPase, as a regulator of axonal regeneration. After sciatic nerve injury in mice, the expression levels of ßPix increase significantly in nerve segments containing regenerating axons. In regrowing axons, ßPix is localized in the peripheral domain of the growth cone. Using ßPix neuronal isoform knockout (NIKO) mice in which the neuronal isoforms of ßPix are specifically removed, we demonstrate that ßPix promotes neurite outgrowth in cultured dorsal root ganglion neurons and in vivo axon regeneration after sciatic nerve crush injury. Activation of cJun and STAT3 in the cell bodies is not affected in ßPix NIKO mice, supporting the local action of ßPix in regenerating axons. Finally, inhibiting Src, a kinase previously identified as an activator of the ßPix neuronal isoform, causes axon outgrowth defects in vitro, like those found in the ßPix NIKO neurons. Altogether, these data indicate that ßPix plays an important role in axonal regrowth during peripheral nerve regeneration.


Assuntos
Axônios , Traumatismos dos Nervos Periféricos , Animais , Camundongos , Regeneração Nervosa , Fatores de Troca de Nucleotídeo Guanina Rho , Neurônios , Cones de Crescimento , Camundongos Knockout
15.
J Cell Sci ; 136(16)2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37534394

RESUMO

The regulation of the intracellular level of ATP is a fundamental aspect of bioenergetics. Actin cytoskeletal dynamics have been reported to be an energetic drain in developing neurons and platelets. We addressed the role of actin dynamics in primary embryonic chicken neurons using luciferase assays, and by measurement of the ATP/ADP ratio using the ratiometric reporter PercevalHR and the ATP level using the ratiometric reporter mRuby-iATPSnFR. None of the methods revealed an effect of suppressing actin dynamics on the decline in the neuronal ATP level or the ATP/ADP ratio following shutdown of ATP production. Similarly, we find that treatments that elevate or suppress actin dynamics do not alter the ATP/ADP ratio in growth cones, the subcellular domain with the highest actin dynamics in developing neurons. Collectively, the data indicate that actin cytoskeletal dynamics are not a significant energy drain in developing neurons and that the ATP/ADP ratio is maintained when energy utilization varies. Discrepancies between prior work and the current data are discussed with emphasis on methodology and interpretation of the data.


Assuntos
Actinas , Cones de Crescimento , Embrião de Galinha , Animais , Actinas/metabolismo , Cones de Crescimento/metabolismo , Citoesqueleto/metabolismo , Metabolismo Energético , Trifosfato de Adenosina/metabolismo
16.
Open Biol ; 13(6): 220359, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37282493

RESUMO

The mechanism of axon growth and guidance is a core, unsolved problem in neuroscience and cell biology. For nearly three decades, our view of this process has largely been based on deterministic models of motility derived from studies of neurons cultured in vitro on rigid substrates. Here, we suggest a fundamentally different, inherently probabilistic model of axon growth, one that is grounded in the stochastic dynamics of actin networks. This perspective is motivated and supported by a synthesis of results from live imaging of a specific axon growing in its native tissue in vivo, together with single-molecule computational simulations of actin dynamics. In particular, we show how axon growth arises from a small spatial bias in the intrinsic fluctuations of the axonal actin cytoskeleton, one that produces net translocation of the axonal actin network by differentially modulating local probabilities of network expansion versus compaction. We discuss the relationship between this model and current views of axon growth and guidance mechanism and demonstrate how it offers explanations for various longstanding puzzles in this field. We further point out the implications of the probabilistic nature of actin dynamics for many other processes of cell morphology and motility.


Assuntos
Actinas , Cones de Crescimento , Cones de Crescimento/fisiologia , Axônios/fisiologia , Neurônios/fisiologia , Citoesqueleto de Actina
17.
J Mater Chem B ; 11(27): 6308-6318, 2023 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-37326438

RESUMO

Long-gap peripheral nerve injury remains a major challenge in regenerative medicine and results in permanent sensory and motor dysfunction. Nerve guidance scaffolds (NGSs) are known as a promising alternative to autologous nerve grafting. The latter, the current "gold standard" in clinical practice, is frequently constrained by the limited availability of sources and the inevitable damage to the donor area. Given the electrophysiological properties of nerves, electroactive biomaterials are being intensively investigated in nerve tissue engineering. In this study, we engineered a conductive NGS compounded of biodegradable waterborne polyurethane (WPU) and polydopamine-reduced graphene oxide (pGO) for repairing impaired peripheral nerves. The incorporation of pGO at the optimal concentration (3 wt%) promoted in vitro spreading of Schwann cells (SCs) with high expression of the proliferation marker S100 protein. In an in vivo study of sciatic nerve transection injury, WPU/pGO NGSs were found to regulate the immune microenvironment by activating macrophage M2 polarization and upregulate growth-associated protein 43 (GAP43) to facilitate axonal elongation. Histological and motor function analysis demonstrated that WPU/pGO NGSs had a neuroprosthetic effect close to that of an autograft, which significantly promoted the regeneration of myelinated axons, reduced gastrocnemius atrophy, and enhanced hindlimb motor function. These findings together suggested that electroactive WPU/pGO NGSs may represent a safe and effective strategy to manage large nerve defects.


Assuntos
Traumatismos dos Nervos Periféricos , Neuropatia Ciática , Animais , Poliuretanos , Cones de Crescimento/metabolismo , Cones de Crescimento/patologia , Nervos Periféricos/fisiologia , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/tratamento farmacológico
18.
Mol Biol Cell ; 34(8): ar83, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37223966

RESUMO

Ena/VASP proteins are processive actin polymerases that are required throughout animal phylogeny for many morphogenetic processes, including axon growth and guidance. Here we use in vivo live imaging of morphology and actin distribution to determine the role of Ena in promoting the growth of the TSM1 axon of the Drosophila wing. Altering Ena activity causes stalling and misrouting of TSM1. Our data show that Ena has a substantial impact on filopodial morphology in this growth cone but exerts only modest effects on actin distribution. This is in contrast to the main regulator of Ena, Abl tyrosine kinase, which was shown previously to have profound effects on actin and only mild effects on TSM1 growth cone morphology. We interpret these data as suggesting that the primary role of Ena in this axon may be to link actin to the morphogenetic processes of the plasma membrane, rather than to regulate actin organization itself. These data also suggest that a key role of Ena, acting downstream of Abl, may be to maintain consistent organization and reliable evolution of growth cone structure, even as Abl activity varies in response to guidance cues in the environment.


Assuntos
Actinas , Cones de Crescimento , Animais , Actinas/metabolismo , Axônios/metabolismo , Drosophila/metabolismo , Cones de Crescimento/metabolismo , Proteínas Proto-Oncogênicas c-abl
19.
J Biol Chem ; 299(5): 104687, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37044214

RESUMO

Axon pathfinding is an essential step in neuronal network formation. Shootin1a is a clutch-linker molecule that is mechanically involved in axon outgrowth and guidance. It was previously shown that concentration gradients of axon guidance molecule netrin-1 in the extracellular environment elicit asymmetrically localized Pak1 kinase-mediated phosphorylation of shootin1a within axonal growth cones, which is higher on the netrin-1 source side. This asymmetric phosphorylation promotes shootin1a-mediated local actin-adhesion coupling within growth cones, thereby generating directional forces for turning the growth cone toward the netrin-1 source. However, how the spatial differences in netrin-1 concentration are transduced into the asymmetrically localized signaling within growth cones remains unclear. Moreover, the protein phosphatases that dephosphorylate shootin1a remain unidentified. Here, we report that protein phosphatase-1 (PP1) dephosphorylates shootin1a in growth cones. We found that PP1 overexpression abolished the netrin-1-induced asymmetric localization of phosphorylated shootin1a as well as axon turning. In addition, we show PP1 inhibition reversed the asymmetrically localized shootin1a phosphorylation within growth cones under netrin-1 gradient, thereby changing the netrin-1-induced growth cone turning from attraction to repulsion. These data indicate that PP1-mediated shootin1a dephosphorylation plays a key role in organizing asymmetrically localized phosphorylated shootin1a within growth cones, which regulates netrin-1-induced axon guidance.


Assuntos
Orientação de Axônios , Proteínas do Tecido Nervoso , Netrina-1 , Proteína Fosfatase 1 , Animais , Camundongos , Axônios/metabolismo , Células Cultivadas , Cones de Crescimento/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Netrina-1/metabolismo , Proteína Fosfatase 1/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
20.
Development ; 150(7)2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-37014062

RESUMO

In the polarity/protrusion model of growth cone repulsion from UNC-6/netrin, UNC-6 first polarizes the growth cone of the VD motor neuron axon via the UNC-5 receptor, and then regulates protrusion asymmetrically across the growth cone based on this polarity. UNC-6 stimulates protrusion dorsally through the UNC-40/DCC receptor, and inhibits protrusion ventrally through UNC-5, resulting in net dorsal growth. Previous studies showed that UNC-5 inhibits growth cone protrusion via the flavin monooxygenases and potential destabilization of F-actin, and via UNC-33/CRMP and restriction of microtubule plus-end entry into the growth cone. We show that UNC-5 inhibits protrusion through a third mechanism involving TOM-1/tomosyn. A short isoform of TOM-1 inhibited protrusion downstream of UNC-5, and a long isoform had a pro-protrusive role. TOM-1/tomosyn inhibits formation of the SNARE complex. We show that UNC-64/syntaxin is required for growth cone protrusion, consistent with a role of TOM-1 in inhibiting vesicle fusion. Our results are consistent with a model whereby UNC-5 utilizes TOM-1 to inhibit vesicle fusion, resulting in inhibited growth cone protrusion, possibly by preventing the growth cone plasma membrane addition required for protrusion.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Cones de Crescimento/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Axônios/metabolismo , Netrinas/metabolismo , Proteínas de Transporte/metabolismo , Receptores de Netrina/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fatores de Crescimento Neural/metabolismo , Moléculas de Adesão Celular/metabolismo
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